Project Summary The transmission of arboviruses from insect hosts to animals and humans results in infections that range from benign and self-limiting to fatal. However, the conserved immunological factors that restrict arbovirus replication and pathogenesis in these diverse hosts are not well understood. We developed an in vivo model system using the free-living nematode, Caenorhabditis elegans, to identify host factors that inhibit infection caused by vesicular stomatitis virus (VSV), a model arbovirus. The inexpensive culture, genetic tractability, and small size of this model organism make it a powerful and convenient system for investigating virus-host interactions. We previously showed that VSV infection of C. elegans animals is ultimately lethal. Nevertheless, nematodes with deficiencies in their antiviral RNA interference (RNAi) response exhibit elevated levels of VSV replication and succumb to infection more rapidly. Therefore, it is evident that the RNAi response is a major antiviral defense mechanism that minimizes viral replication and pathogenesis in C. elegans. However, it is unclear if nematodes encode additional mechanisms to restrict arbovirus replication. Here we propose to use our VSV model to identify conserved host factors that mediate resistance to infection in both nematodes and in humans. To discover host factors that may interact with VSV, we conducted studies to identify nematode proteins that physically associate with the VSV nucleocapsid (N) protein, the main structural component of the virus particle. We hypothesized that some of these interacting proteins may be antiviral host factors that either target the VSV N protein or are themselves inhibited by VSV N as part of a viral strategy to subvert host immune responses. Thus far, we have identified four nematode factors with putative VSV N interactions that display antiviral properties in VSV infections of adult nematodes. Importantly, our preliminary results indicate that these factors do not function in the antiviral RNAi pathway, suggesting that they have independent mechanisms of restricting arbovirus infection. Furthermore, we found the human counterparts (orthologs) of these nematode proteins to also exhibit antiviral activities against VSV in human cell cultures. These results suggest that our unique approach may uncover conserved facets of virus-host interplay. Our proposed studies have the following specific aims: 1) use the C. elegans model to identify additional host factors that inhibit arbovirus replication; 2) determine if human orthologs of the identified nematode factors also contribute to arbovirus restriction; and 3) develop tools for further characterization of these virus-host interactions. Our long-term goal is to use this model system to define key eukaryotic innate immune mechanisms that restrict arbovirus replication. Identifying such interactions may lead to new strategies for the treatment of human arbovirus infections. This is particularly important given the global public health threat posed by arboviruses and our current lack of specific therapeutics for arboviral disease.